Most membrane fusion reactions in eukaryotic cells are executed by membrane-bridging SNARE complexes. The formation of these complexes generally requires that four different SNARE proteins, anchored in two different membranes, undergo a coupled folding and assembly reaction during which the SNARE motifs zipper up into a parallel four-helix bundle. This complicated process is inefficient in vitro, and is certain to be even more challenging in vivo, where it must compete with the formation of various non-cognate and off-pathway SNARE complexes. Consequently, we hypothesize that most SNARE complex assembly reactions in the cell are orchestrated by a set of `topologically aware' chaperones called multisubunit tethering complexes (MTCs). These highly-conserved nanomachines interact directly with virtually all of the proteins (including the SNAREs) implicated in membrane tethering and fusion. We furthermore propose that the key task of catalyzing four-helix bundle formation falls to the Sec1/Munc18 (SM) proteins, working together with ? and sometimes as integral subunits of ? the MTCs. Therefore, the overarching goal of this proposal is to achieve an improved structural and mechanistic understanding of MTC and SM function in the assembly of fusogenic SNARE complexes. In Aim 1, we will conduct structural studies of the homotypic fusion and vacuole protein sorting (HOPS) complex, a well-studied MTC required for fusion at late endosomes and lysosomes/vacuoles. High-resolution cryo- electron microscopy will be used to elucidate the structure of the HOPS complex. In order to elucidate how HOPS organizes SNAREs for assembly, we will also determine crystal structures of complexes between HOPS subunits and SNARE N-terminal regulatory domains. These structures will then serve as blueprints for in vivo and in vitro functional studies. In Aim 2, we will elucidate the detailed mechanism by which a subunit of the HOPS complex, the SM protein Vps33, catalyzes SNARE assembly. These studies will encompass fluorescence-based biochemical assays, crystal structures of SNARE assembly intermediates, reconstituted proteoliposome-based fusion assays, and single-molecule optical tweezers experiments. In Aim 3, we will expand these studies to other SM proteins, using the same suite of experimental approaches to test the generality of our Vps33-derived mechanistic model. These experiments will also afford us an opportunity to evaluate the extent to which the catalytic activity of SM proteins is under regulatory control. Finally, in Aim 4, we will turn our attention to the simplest known MTC, the Dsl1 complex. Using X-ray crystallography and in vivo imaging, we will rigorously test the role of the Dsl1 complex in vesicle tethering and tethering-triggered vesicle uncoating.